Author

Date of Award

Degree Type

Degree Name

Department

Medicinal Chemistry and Molecular Pharmacology

First Advisor

Ryan M Drenan

Committee Member 1

Val J Watts

Committee Member 2

Gregory H Hockerman

Committee Member 3

Edward L Bartlett

Abstract

Tobacco addiction is a serious threat to public health in the United States and abroad, and development of new therapeutic approaches is a major priority. Nicotine, the primary psychoactive compound in tobacco smoke, activates and/or desensitizes nicotinic acetylcholine receptors (nAChRs) throughout the brain. nAChRs in ventral tegmental area (VTA) dopamine (DA) neurons are crucial for the rewarding and reinforcing properties of nicotine. Nicotine causes cellular changes in VTA DA neurons, including the enhancement of AMPA receptor (AMPAR) function. This enhancement sensitizes the VTA to excitatory input and promotes drug seeking in animal models. However, which nAChR subtype(s) are responsible for initiating these cellular changes is poorly understood. nAChRs containing the α6 subunit (α6* nAChRs) are highly and selectively expressed in DA neurons in the VTA. Therefore, we hypothesized that activation of α6* nAChRs is sufficient to enhance AMPAR function on the surface of VTA DA neurons. To test this, we studied mice expressing hypersensitive, gain-of-function α6 nAChRs (α6L9S mice). We found that low concentrations of nicotine could act selectively through α6* nAChRs to enhance the function of AMPARs on the surface of VTA DA neurons. Through pretreatment with pharmacological inhibitors, we found that NMDA receptors, as well as Ca2+/calmodulin dependent protein kinase II, are also required for this effect. We subsequently expanded these studies to include alcohol because of the high rate of tobacco and alcohol co-abuse. Just as with nicotine, we found that low concentrations of ethanol were sufficient to enhance AMPAR function on VTA DA neurons of α6L9S mice. Because ethanol and nicotine both modulate AMPAR function in a manner involving α6* nAChRs, we tested the hypothesis that low concentrations of ethanol and nicotine combine to modulate AMPAR function. Remarkably, co-incubation of α6L9S brain slices in concentrations of ethanol and nicotine that are sub-threshold when incubated alone resulted in robust enhancement of AMPAR function. Within the VTA, α6 nAChR subunits form nAChRs with and without the α4 nAChR subunit. Therefore, we studied the contribution of α4 nAChR subunits to nicotine-elicited changes in VTA synaptic plasticity. To address this, we removed α4 nAChR subunits from the VTA of adult mice by injecting viral vectors directing expression of Cre recombinase into the VTA of mice with loxP sites flanking the α4 subunit gene. We found that nicotine no longer increases AMPAR function when α4 nAChR subunits are removed from the VTA, indicating a role of nAChRs that contain both α4 and α6 nAChR subunits in VTA synaptic plasticity. Interestingly, we also saw that removing α4 subunits from the VTA of adult mice increases the excitability of VTA DA neurons. We hypothesized that removal of α4* nAChRs from GABAergic neurons in the VTA results in less tonic inhibition of VTA DA neurons. To test this we measured spontaneous inhibitory postsynaptic currents (IPSCs) on VTA DA neurons. Indeed, we saw that the instantaneous frequency of IPSCs was significantly reduced when α4 nAChR subunits are removed from the VTA. Overall, these studies highlight the importance of α4α6* nAChRs in the initiation of cellular changes that play a role in addiction to nicotine, suggesting α4α6* nAChRs may be a promising target for future smoking cessation pharmacotherapies.